Clostridium botulinum neurotoxins are produced as progenitor toxins whose specific toxicity increases when acted upon by enzymes such as trypsin. Among the enzymes which determine the proteolytic behavior of some C. botulinum cultures (types A, B, and F) lack these enzymes. Thus, trypsinization of nonproteolutic culture filtrates result in relatively high toxicity increases while none or low toxicity increases occur with filtrates of proteolytic cultures. The proposed work seeks to identify and isolate the botulinal enzymes which activate toxin and to determine the bonds whose cleavage results in the high toxicity of botulinal toxin. Fully activated toxins are proteins (mol wt 150,000) in which disulfides are important to hold together constituent polypeptide units of mol wt 100,000 and 50,000. Attempts are being made to separate these units without using protein denaturants so that the toxicity and antigenicity of these units can be studied.